Bottom Line:
Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions.We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population.We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

Methodology: A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal findings: Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions: We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

pone-0003319-g004: Natalizumab does not alter the suppressive capacity of CD4+CD25highCD127low Tregs and does not restore impaired Treg function in MS.(A) CFSE-based suppression assays using fluorescence activated cell sorted T cells derived from the peripheral blood of healthy donors were conducted in the presence or absence of Natalizumab (20 µg/ml). The proliferation of responder T cells (CD4+CD25−) was suppressed in the presence of Tregs as analyzed by flow cytometry, however, the suppressive capacity of Tregs remained unaffected by the mAb. One out of 5 representative experiments is shown, the percentage of proliferating cells is indicated in the diagram. (B) Suppressive capacity of serially analyzed T cells derived from healthy donors (n = 5) and Natalizumab treated patients (n = 5 for each time point except month 6: n = 3) before (M0), 1 month (M1), 3 months (M3) and 6 months after initiation of therapy. Bars show the proliferation of responder cells (CD4+CD25−) in the presence of Tregs (1∶10 Treg to T effector ratio) after normalization to the proliferation of responder cells in the presence of an equal number of non-regulatory T cells. * p<0,05; Significant differences in suppression are observed comparing HD and RRMS Tregs at any time point during the treatment period, as exemplarily depicted for M0. (C) Percentage of proliferating T effector cells derived from healthy donors (n = 5) that were challenged by immature or mature dendritic cells (DC : T cell ratio 1∶5) in the presence or absence of Natalizumab (20 µg/ml). (D) In vitro addition of Natalizumab (20 µg/ml) does not affect Treg mediated suppression of T cell proliferation in response to immature allogeneic DC as analyzed in CFSE suppression assays. Data were normalized to the proliferation of T cells in response to DC in the presence of an equal number of non-regulatory T cells. (E) Proliferation of responder cells from healthy controls (HD, n = 4), and RRMS patients before (M0, n = 4), 1 month (M1, n = 4) and six months (M6, n = 4) after initiation of Natalizumab treatment. Normalization as in (D).

Mentions:
In contrast to Tregs derived from the peripheral blood of healthy donors, MS patient derived Tregs showed very poor suppression of autologous T cell proliferation as a baseline (31% versus 4% mean suppression, at a 1∶10 Treg to responder ratio; n = 5 for healthy donors and MS patients respectively). Addition of Natalizumab to the αCD3/αCD28 microbead-based suppression assays showed no significant influence on the suppressor function of Foxp3+ regulatory T cells (experiments performed with Tregs derived from healthy donors; one out of 5 representative experiment is shown, Figure 4a). Various concentrations of Natalizumab (0,5 µg/ml up to 40 µg/ml) and titration of suppressor∶effector ratios in modification of the assay conditions yielded comparable results (data not shown). We tested the effect of Natalizumab treatment on the suppressive function of Tregs in ex vivo αCD3/αCD28 microbead-based suppression experiments. In line with our in vitro data, treatment with Natalizumab did not alter the dysfunctional suppressive capacity of T regulatory cells in patients with RRMS. Over 6 months of treatment, highly pure CD4+CD25highCD127low regulatory cells remained poor suppressors (n = 5, Figure 4b). In addition to bead-based suppression assays we used allogeneic immature and mature DC to challenge T effector cells. Since VLA-4 is part of the immunological synapse and has been suggested to be involved in costimulation [11]–[13] we first investigated whether Natalizumab influences the outcome of DC-T cell encounters by modulating T cell proliferation. However, Natalizumab (20 µg/ml) did not impact the percentage of proliferating T cells in response to immature DC (23,96+/−10,57 vs. 24,58+/−14,02) or mature DC (39,324+/−14,77 vs. 39,178+/−16,67) as determined by CFSE dilution assays (n = 5, Figure 4c). APC-based suppression assays revealed that CD4+CD127lowCD25high Treg cells derived from healthy donors strongly suppressed the proliferation of T effector cells challenged by allogeneic DC, as exemplarily depicted for immature DC (Figure 4c). VLA-4 blockade by addition of Natalizumab (20 µg/ml) did not affect the Treg mediated suppression in the presence of immature DC (Figure 4d). Similar results were obtained when LPS matured DC were used instead of immature DC (data not shown). Further analysis confirmed that Tregs derived from MS patients remained dysfunctional in this APC-based suppression assay (Figure 4e, n = 4). Ex vivo experiments using T effectors and T regulatory cells derived from Natalizumab treated patients before and after initiation of therapy (month 1 and 6) did not reveal a significant effect on the suppressive capacity of CD4+CD127lowCD25high Treg in the presence of allogeneic immature DC, albeit a trend towards a regain of function could be noted (Figure 4e, n = 4).

pone-0003319-g004: Natalizumab does not alter the suppressive capacity of CD4+CD25highCD127low Tregs and does not restore impaired Treg function in MS.(A) CFSE-based suppression assays using fluorescence activated cell sorted T cells derived from the peripheral blood of healthy donors were conducted in the presence or absence of Natalizumab (20 µg/ml). The proliferation of responder T cells (CD4+CD25−) was suppressed in the presence of Tregs as analyzed by flow cytometry, however, the suppressive capacity of Tregs remained unaffected by the mAb. One out of 5 representative experiments is shown, the percentage of proliferating cells is indicated in the diagram. (B) Suppressive capacity of serially analyzed T cells derived from healthy donors (n = 5) and Natalizumab treated patients (n = 5 for each time point except month 6: n = 3) before (M0), 1 month (M1), 3 months (M3) and 6 months after initiation of therapy. Bars show the proliferation of responder cells (CD4+CD25−) in the presence of Tregs (1∶10 Treg to T effector ratio) after normalization to the proliferation of responder cells in the presence of an equal number of non-regulatory T cells. * p<0,05; Significant differences in suppression are observed comparing HD and RRMS Tregs at any time point during the treatment period, as exemplarily depicted for M0. (C) Percentage of proliferating T effector cells derived from healthy donors (n = 5) that were challenged by immature or mature dendritic cells (DC : T cell ratio 1∶5) in the presence or absence of Natalizumab (20 µg/ml). (D) In vitro addition of Natalizumab (20 µg/ml) does not affect Treg mediated suppression of T cell proliferation in response to immature allogeneic DC as analyzed in CFSE suppression assays. Data were normalized to the proliferation of T cells in response to DC in the presence of an equal number of non-regulatory T cells. (E) Proliferation of responder cells from healthy controls (HD, n = 4), and RRMS patients before (M0, n = 4), 1 month (M1, n = 4) and six months (M6, n = 4) after initiation of Natalizumab treatment. Normalization as in (D).

Mentions:
In contrast to Tregs derived from the peripheral blood of healthy donors, MS patient derived Tregs showed very poor suppression of autologous T cell proliferation as a baseline (31% versus 4% mean suppression, at a 1∶10 Treg to responder ratio; n = 5 for healthy donors and MS patients respectively). Addition of Natalizumab to the αCD3/αCD28 microbead-based suppression assays showed no significant influence on the suppressor function of Foxp3+ regulatory T cells (experiments performed with Tregs derived from healthy donors; one out of 5 representative experiment is shown, Figure 4a). Various concentrations of Natalizumab (0,5 µg/ml up to 40 µg/ml) and titration of suppressor∶effector ratios in modification of the assay conditions yielded comparable results (data not shown). We tested the effect of Natalizumab treatment on the suppressive function of Tregs in ex vivo αCD3/αCD28 microbead-based suppression experiments. In line with our in vitro data, treatment with Natalizumab did not alter the dysfunctional suppressive capacity of T regulatory cells in patients with RRMS. Over 6 months of treatment, highly pure CD4+CD25highCD127low regulatory cells remained poor suppressors (n = 5, Figure 4b). In addition to bead-based suppression assays we used allogeneic immature and mature DC to challenge T effector cells. Since VLA-4 is part of the immunological synapse and has been suggested to be involved in costimulation [11]–[13] we first investigated whether Natalizumab influences the outcome of DC-T cell encounters by modulating T cell proliferation. However, Natalizumab (20 µg/ml) did not impact the percentage of proliferating T cells in response to immature DC (23,96+/−10,57 vs. 24,58+/−14,02) or mature DC (39,324+/−14,77 vs. 39,178+/−16,67) as determined by CFSE dilution assays (n = 5, Figure 4c). APC-based suppression assays revealed that CD4+CD127lowCD25high Treg cells derived from healthy donors strongly suppressed the proliferation of T effector cells challenged by allogeneic DC, as exemplarily depicted for immature DC (Figure 4c). VLA-4 blockade by addition of Natalizumab (20 µg/ml) did not affect the Treg mediated suppression in the presence of immature DC (Figure 4d). Similar results were obtained when LPS matured DC were used instead of immature DC (data not shown). Further analysis confirmed that Tregs derived from MS patients remained dysfunctional in this APC-based suppression assay (Figure 4e, n = 4). Ex vivo experiments using T effectors and T regulatory cells derived from Natalizumab treated patients before and after initiation of therapy (month 1 and 6) did not reveal a significant effect on the suppressive capacity of CD4+CD127lowCD25high Treg in the presence of allogeneic immature DC, albeit a trend towards a regain of function could be noted (Figure 4e, n = 4).

Bottom Line:
Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions.We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population.We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

Methodology: A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal findings: Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25(high)CD127(low)Foxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions: We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.